Method for culturing T lineage precursor cells under conditions of high oxygen concentration

ABSTRACT

The present invention provides culture methods of T lineage precursor cells comprising a step of culturing the T lineage precursor cells under a condition that a dissolved oxygen concentration in the nutrient medium is higher than a dissolved oxygen concentration in the nutrient medium when the medium is in contact with normal atmospheric air. According to the methods of the present invention, by using a submerged (suspension) culture which is a general method for culturing cells or tissues, the derivation of matured T cell(s) differentiated from T precursor cells in terms of their phenotypes of differentiation antigens and of their functions is realized to be accomplished.

This application is a continuation divisional of application Ser. No.07/983,514, filed as PCT/JP92/00829 Jul. 1, 1992, now U.S. Pat. No.5,476,780, the entire contents of which are hereby incorporated byreference.

TECHNICAL FIELD!

The present invention relates to a method for culturing T lineageprecursor cells, which enables derivation and generation of matured Tcells differentiated from the T lineage precursor cells. The inventionfurther relates to: T cells obtained by using this method.

BACKGROUND ART!

The earliest of T cells exist mainly in bone marrow or fetal liver, as aform of lymphoid stem cells differentiated from hematopoietic stem cellshaving an ability to differentiate into multilineage cells. Theseearliest stem cells emigrate to thymus and mature into T cells through aseries of differentiation stages. The differentiation and maturation ofthe T precursor cells are greatly under the influence of thymic stromalcells. After T precursor cells enter into the thymic cortex, the Tprecursor cells differentiate and mature into T cells through rapid celldivision and proliferation under the control of adhesion with stromalcells comprising mainly thymic epithelial cells which participate indifferentiation and maturation of the T precursor cells, adhesion withmyeloid cells such as macrophages, and humoral factors producedtherefrom. After undergoing several differentiation stages thymocytesflow out to the bloodstream from the cortico-medullary junction ofthymus, and emigrate to peripheral lymphoid organs. The emigratedthymocytes, i.e., T cells, further differentiate and mature therein.

To study the detailed process of differentiation and maturation of the Tprecursor cells, a culture system capable of achieving in-vitroproliferation, differentiation, and maturation of T cells is required.

On the other hand, it is considered that abnormality of differentiationor of functions of T cells due to immunization with infectious bacteriaand/or an foreign antigen is one great cause of allergic diseases orvarious autoimmune diseases. (e.g., Animesh A. et al, Science, vol. 248,1380-1388, 1990; Irun R. Cohen et al, Immunology Today, vol. 12, No. 4,105-110, 1991; and Lars Klareskog et al, Immunological Reviews, vol.118, 285-310, 1990).

At present, as therapeutic drugs for such diseases, non-specificanti-inflammatory agents and immunosuppressants of T cell functions areused as suppressors. However, essential cure of the diseases has notbeen achieved due to their non-specificity.

To overcome these problems, extensive studies on vaccine therapy andserum vaccine therapy have been made, comprising respectively (1)specifying an antigen which causes disease and applying the antigen, and(2) specifying said antigen and applying suppressive factors specificfor the diseases which are represented by antibodies against theantigen. A cell vaccine therapy using T cells whose antigenspecificities has been proved is considered to be an effectivetherapeutic method for diseases which is assumed to be caused byabnormal differentiation and functional abnormality of T cells (e.g.,Jose C. Gutierrez-Ramos et al, Nature, vol. 346, 271-274, 1990 and KoheiOta et al, Nature, vol. 346, 183-187, 1990). However, methods capable ofderiving T cells having appropriate antigen specificity in vitro arelimited to a particular method.

In fact, although a submerged (suspension) culture in which cells ortissues are submerged or suspended in a nutrient medium is well known asa method generally used for culture of cells or tissues, it isimpossible to differentiate and mature T precursor cells into T cells inaccordance with such a conventional method.

At present, as the sole culture method capable of achievingdifferentiation and maturation of the T precursor cells, only aso-called "afloat culture" is known, in which a fetal thymus is culturedon an interface between the gas phase and the nutrient medium underatmospheric air whose carbon dioxide concentration is controlled to 5%(e.g., Jenkinsen E. J. et al, Eur. J. Immunology, vol. 12, 583, 1982 andOsamu Mazda et al, J. Exp. Med., vol. 173, 539-547, 1991). However, thismethod requires special tools or apparatuses, and differentiation ofcells on the liquid surface side is insufficient. In addition to thesedrawbacks, this method is not suitable for the culture of the Tprecursor cells accompanying mass proliferation of cells. Therefore, itis difficult to perform a large scale culture.

Strong demand has, therefore, arisen for developing a culture methodcapable of realizing and achieving derivation of matured T cellsdifferentiated from T precursor cells in terms of their phenotypes ofdifferentiation antigens and of their functions, by using the submerged(suspension) culture as a general method of culturing cells or tissues.

Japanese Unexamined PCT Application No. 3-504329 discloses a method forproliferating a human B blastic cell line by raising the dissolvedoxygen concentration in the culture medium by steadily supplying adesired amount of oxygen into the medium placed in a sealed vessel,through a silicon pipe connected to a gas bomb sealed with a gas mixturecontaining a desired amount of oxygen. In said culture according to themethod, the proliferation of the human B blastic cell line is found butdifferentiation and maturation of the cells is not achieved. The presentinvention is capable of achieving proliferation of T precursor cells,and of differentiation and maturation of them in terms of theirphenotypes of differentiation antigens and of their functions, by usinga simpler culture method than that disclosed in above application undera higher concentration of dissolved oxygen in the medium.

DISCLOSURE OF INVENTION!

It is an object of the present invention to provide a culture methodcapable of realizing and achieving derivation of matured T cells,differentiated from T precursor cells in terms of their phenotypes ofdifferentiation antigens and of their functions, by using the submerged(suspension) culture as a general method of culturing cells or tissues.

In order to achieve the object, the present inventors made extensivestudies on a method of in-vitro culture of T lineage precursor cells andfound that matured T cells, differentiated from T lineage precursorcells in terms of their phenotypes of differentiation antigens or theirfunctions, could very be efficiently and easily derived and generated byculturing T lineage precursor cells under a condition wherein thedissolved oxygen concentration in a nutrient medium was higher than thatin the normal atmospheric air, i.e., under a condition of high oxygenconcentration, thereby achieving the present invention.

That is, a method of culturing T lineage precursor cells according tothe present invention comprises a step of culturing the T lineageprecursor cells in a nutrient medium under condition such that thedissolved oxygen concentration in the nutrient medium is higher thanthat present in the normal atmospheric air.

According to the present invention, by using a submerged (suspension)culture well known as a general method of culturing cells or tissues,matured T cells, normally differentiated from T precursor cells in termsof their phenotypes of differentiation antigens or their functions, canbe derived and the T precursor cells can be proliferated. In addition,according to the present invention, the culture can be very easilyperformed without using special tools or apparatuses, and a large scaleculture can be performed.

The present invention is described in detail below.

The terms "T lineage precursor cells", "T precursor cells", "tissue(s)containing T precursor cells", "interstitial cells of lymphoid tissues","nutrient medium", "dissolved oxygen concentration in nutrient medium",and "matured T cells differentiated in terms of their phenotypes ofdifferentiation antigens or their functions" are defined as follows.

a) "T lineage Precursor cells"

The "T lineage precursor cells" used in the present invention mean "Tprecursor cells", "tissue(s) containing T precursor cells", or a"combination of the T precursor cells and the interstitial cells oflymphoid cells". The term "T lineage precursor cells" can includehematopoletic stem cells.

The "T lineage precursor cells" can be collected from any tissue(s)having T precursor cells regardless of the species, sex, and age of thedonor. Preferably, the T lineage precursor cells can be collected from amammal such as a rat, a mouse, a rabbit, a horse, or a cow. Morepreferably, T lineage precursor cells are collected from a fetus orneonate of said mammal, and can be collected from a human.

A population of cells participating in the immune response isgenerically called immunocompetent cells. Among the immunocompetentcells, T- and B-lineage lymphoid cells are related to antigen memory andplay principal roles of immune responses. The present invention relatesto the T-lineage cells. The origins of the T-lineage cells are lymphoidstem cells differentiated from hematopoietic stem cells existing mainlyin bone marrow or fetal liver, and the lymphoid stem cells develop intoT lineage stem cells through differentiation. The T lineage stem cellsemigrate to the thymus and mature to T cells having an ability ofplaying a role of cellular immunity through respective stages ofdifferentiation accompanying proliferation.

The term "T precursor cells" used in the present invention means any Tlineage cells at any differentiation stage in a series ofdifferentiation and maturation processes from lymphoid stem cells tomatured T cells, as described above. For example, the population of"fetal thymocyte(s)" referred to in this specification contains a largenumber of T lineage stem cells which have emigrated to thymus, and isvery useful as a source of "T precursor cells". The phrase "tissue(s)containing T precursor cells" means tissue(s) containing said "Tprecursor cells". Specifically, the tissue(s) mean tissue(s) or theirfragments originated from, e.g., thymus, bone marrow or liver, and morepreferably thymus or bone marrow. The "interstitial cells of lymphoidtissues" mean a cell lineage (e.g., thymic epithelial cells,fibroblasts, and intercellular matrix), except for the "T precursorcells", which supports and surrounds the "T precursor cell" in the"tissue(s) containing the T precursor cells". According to the presentinvention, "T precursor cells" and "interstitial cells of lymphoidtissues" which are respectively obtained from the same or differentindividuals of the same or different animal species can be combined topractice the present invention. For example, the present invention ispracticed by a combination of fetal thymocytes and thymic interstitialcells. More specifically, the present invention is carried out by acombination of human T precursor cells and mouse thymic interstitialcells, a combination of mouse T precursor cells and human thymicinterstitial cells, a combination of human T precursor cells and ratthymic interstitial cells, or a combination of rat T precursor cells andhuman thymic interstitial cells.

b) "Nutrient Medium"

The "nutrient medium" used in the present invention is employed todifferentiate, proliferate, mature, maintain, or preserve the T lineageprecursor cells defined in a). Any conventional medium for culturingcells can be used. An example of the nutrient medium is an RPMI-1640medium or an MEM medium. The nutrient medium can contain, as elementalcomponents, sodium, potassium, calcium, magnesium, phosphorus, chlorine,amino acid(s), vitamin(s), hormone(s), antibiotics, serum, or otherchemical components depending on application purposes.

The nutrient medium can contain a cytokine such as interleukin-1,interleukin-2, interleukin-3, interleukin-4, interleukin-5,interleukin-6, interleukin-7, interleukin-8, interleukin-9,interleukin-10, interleukin-11, interleukin-12, TNF (Tumour NecrosisFactor), γ-interferon, a granulocyte macrophage colony-stimulatingfactor (GM-CSF), a granulocyte colony-stimulating factor (G-CSF), or amacrophage colony-stimulating factor (M-CSF), depending on applicationpurposes. By using nutrient medium containing an antigen which inducesinfectious diseases due to such as a hepatitis virus, an HIV (HumanImmunodeficiency Virus) and so on, allergic diseases and autoimmunediseases, T cell vaccines for suppressing and treating the symptoms ofinfectious diseases, inflammation, allergy, and autoimmune diseasesinduced by the antigens can be derived and generated.

c) "Dissolved oxygen Concentration in Nutrient Medium"

The term "dissolved oxygen concentration in nutrient medium" used in thepresent invention means the concentration of oxygen dissolved in anutrient medium (liquid phase) for culturing the T lineage precursorcells defined in a). The phrase "dissolved oxygen concentration innutrient medium under normal atmospheric air" indicates the dissolvedoxygen concentration in a nutrient medium saturated with naturalatmospheric air which is not artificially modified. Therefore, forexample, "the dissolved oxygen concentration in a nutrient medium underthe condition that the total pressure of the gas phase contacting thenutrient medium is 1 atm, that the oxygen partial pressure of the gasphase is 0.4 atm, and that the temperature is 37° C." indicates thatsaid dissolved oxygen concentration is twice of that of normalatmospheric air, namely, 1 atm atmospheric air, in accordance withHenry's law which says that the solubility of gas at constanttemperature is proportional to its partial pressure as far as thepressure is not so high.

As a means for raising the dissolved oxygen concentration in the medium,any means can be used if the means has a capacity for supplying oxygento raise the amount of oxygen dissolving in the nutrient medium. Inparticular, it is preferable to substantially raise the dissolved oxygenconcentration in the nutrient medium (liquid phase) by raising theoxygen concentration in the gas phase contacting with the nutrientmedium (liquid phase) within the range of 40 vol % through 95 vol %,preferably 60 vol % through 95 vol %.

The oxygen concentration is expressed in different units such as %, atm(atmosphere), mmHg, and M (mol) herein. Any unit of the oxygenconcentrations expressed can be converted into any other. The oxygenconcentration in the present invention need not be expressed in aspecific unit, as a matter of course.

Since the solubility of oxygen is proportional to the partial pressureof oxygen of the gas phase, it is easily understood for the skilled inthe art that the dissolved oxygen concentration in the medium can beraised by slightly elevating the pressure of the gas phase even if theoxygen concentration of the gas phase is 60 vol % or less, or 40 vol %or less.

In case of necessity of replacing a part of the medium during theculture, it is preferable to control, in advance, the dissolved oxygenconcentration in a medium for replacement to the same dissolved oxygenconcentration in the former medium. Alternatively, the dissolved oxygenconcentration in the medium for replacement can be controlled by usingair flow containing a high amount of oxygen immediately after thereplacement.

d) "Matured T cells Differentiated in Terms of their Phenotypes ofDifferentiation Antigens or their Functions"

T lineage stem cells differentiated and developed from lymphoid stemcells derived from hematopoietic stem cells proliferate and maturethrough each stage of differentiation and maturation under the controlof adhesion with the thymic interstitial cells and of endogenous humoralfactors.

Each stage of differentiation and maturation can be distinguished andclassified by the phenotypes based on difference of kinds ofdifferentiation antigens expressed on the cell surface, or by biologicalfunctions such as abilities of antigen specific proliferation, B cellactivation, cytotoxicity and immunosuppression.

By applying the method of the present invention, it is possible torealize and achieve all stages of differentiation and maturation invitro using the "T lineage precursor cells" defined in a). The term"matured T cells differentiated in terms of their phenotypes ofdifferentiation antigens or their functions" means T lineage cells insaid any stage of differentiation and maturation.

BRIEF DESCRIPTION OF DRAWINGS!

FIG. 1 is a graph showing a correlation between the number of recoveredviable cells and the dissolved oxygen concentration in a nutrient mediumunder each culture condition. Each data point represents culture at adifferent gas phase oxygen concentration. From left to right: gas phaseoxygen concentration of 20% (corresponds to dissolved oxygenconcentration of about 143 mmHg); gas phase oxygen concentration of 40%(corresponds to dissolved oxygen concentration of about 214 mmHg); gasphase oxygen concentration of 60% (corresponds to dissolved oxygenconcentration of about 333 mmHg); gas phase oxygen concentration of 40%(corresponds to dissolved oxygen concentration of about 451 mmHg).

FIGS. 2 to 7 are graphs showing the distributions of the followingvarious types of cells, which is distinguished on the basis of cellsizes determined by using a flow cytometer, wherein relativecomplexities of the cells based on side-light scatter are plotted alongthe ordinates and relative cell sizes based on forward-light scatter areplotted along the abscissas; That is:

FIG. 2 is a graph showing the cell distribution of mouse matured Tcells,

FIG. 3 is a graph showing the cell distribution of mouse fetalthymocytes immediately after these thymocytes are collected,

FIG. 4 is a graph showing the cell distribution after mouse fetalthymuses are cultured by an afloat culture at a 20% oxygenconcentration,

FIG. 5 is a graph showing the cell distribution after mouse fetalthymuses are cultured by a submerged culture at a 5% oxygenconcentration,

FIG. 6 is a graph showing the cell distribution after mouse fetalthymuses are cultured by a submerged culture at a 20% oxygenconcentration, and

FIG. 7 is a graph showing the cell distribution after mouse fetalthymuses are cultured by a submerged culture at a 60% oxygenconcentration;

FIGS. 8 to 15 are graphs showing the cell distributions showing theexpression level of CD4 and/or CD8, each of which is a differentiationantigen on mouse T cell membranes. These expression levels aredistinguished by a fluorescence-linked antibody-mediated analysis usinga flow cytometer, wherein the expression level of CD4 is plotted alongthe ordinates and the expression level of CD8 is plotted along theabscissas; that is:

FIG. 8 is a graph showing the cell distribution which represents theexpression level of CD4 and/or CD8 of mouse matured T cells,

FIG. 9 is a graph showing the cell distribution which represents theexpression level of CD4 and/or CD8 of mouse fetal thymocytes immediatelyafter these thymocytes are collected,

FIG. 10 is a graph showing the cell distribution which represents theexpression level of CD4 and/or CD8 after the mouse fetal thymuses arecultured by an afloat culture at a 20% oxygen concentration,

FIG. 11 is a graph showing the cell distribution which represents theexpression level of CD4 and/or CD8 after the mouse fetal thymuses arecultured by a submerged culture at a 5% oxygen concentration,

FIG. 12 is a graph showing the cell distribution which represents theexpression level of CD4 and/or CD8 after the mouse fetal thymuses arecultured by a submerged culture at a 20% oxygen concentration, and

FIG. 13 is a graph showing the cell distribution which represents theexpression level of CD4 and/or CD8 after the mouse fetal thymuses arecultured by a submerged culture at a 40% oxygen concentration.

FIG. 14 is a graph showing the cell distribution which represents theexpression level of CD4 and/or CD8 after the mouse fetal thymuses arecultured by a submerged culture at a 60% oxygen concentration.

FIG. 15 is a graph showing the cell distribution which represents theexpression level of CD4 and/or CD8 after the mouse fetal thymuses arecultured by a submerged culture at an 80% oxygen concentration.

FIG. 16 is a graph showing the proliferation degree of ConA-reactive,functional T cells measured by using a tritium-labeled thymidine (³H-TdR) incorporation test, wherein the cellular incorporation amounts(cpm) of ³ H-TdR as an index of DNA synthesis accompanying in T cellproliferation are plotted along the ordinate, and the dilution rates ofthe number of cells per a cell population of thymus after the cultureare plotted along the abscissa. -: afloat culture at oxygenconcentration of 20%, ∘-∘: submerged culture at oxygen concentration of5%, ▪-▪: submerged culture at oxygen concentration of 20%, □-□:submerged culture at oxygen concentration of 60%.

FIG. 17 is a graph showing the proliferatrion degree of allo-reactive,functional T cells measured by using a tritium-labeled thymidine (³H-TdR) incorporation test, wherein the cellular incorporation amounts(cpm) of ³ H-TdR as an index of DNA synthesis accompanying in T cellproliferation are plotted along the ordinate, and the dilution rates ofthe number of cells per a cell population of thymus after the cultureare plotted along the abscissa. -: afloat culture at oxygenconcentration of 20%, ∘-∘: submerged culture at oxygen concentration of5%, ▪-▪: submerged culture at oxygen concentration of 20%, □-□:submerged culture at oxygen concentration of 60%.

FIG. 18 is a graph showing the cell distribution which represents theexpression level of CD4 and/or CD8 after mouse thymic interstitial cellsnot containing mouse fetal thymocytes is cultured by a submerged cultureat a 60% oxygen concentration.

FIG. 19 is a graph showing the cell distribution which represents theexpression level of CD4 and/or CD8 after individually collected mousethymic interstitial cells and 300 mouse fetal thymocytes are cultured bya submerged culture at a 60% oxygen concentration.

FIG. 20 is a graph showing the cell distribution which represents theexpression level of CD4 and/or CD8 of mouse matured T cells. and

FIG. 21 is a graph showing a correlation between the oxygenconcentration in the gas phase contacting with the nutrient medium andthe dissolved oxygen concentration in the nutrient medium. Carbondioxide was present in each gas mixture at a constant ratio of 5%.Oxygen in the gas phase varied from 0 to 95%, with the balance of thegas composition being nitrogen. Note that 20% oxygen is the ratio ofoxygen in normal atmospheric air.

BEST MODE OF CARRYING OUT THE INVENTION!

The embodiments of the present invention are described in detail by wayof its examples. The present invention is not limited to the followingexamples, as a matter of course.

EXAMPLE 1

<1-1> Collection of Mouse Thymus

Thymuses were aseptically collected by an ordinary method from 6-weekold C57BL/6 mice (B6 young mice) and 15-day fetuses of B6 mice(available from Nippon SLC Corp.) obtained from time-mated C57BL/6 mice(B6 pregnant mice).

<1-2> Collection of Mouse Thymocytes and Counting

The thymuses of the young mice and the mouse fetal thymuses (FT), whichwere collected in 1-1, were gently minced with stainless steel mesh toafford young mouse thymocytes and mouse fetal thymocytes.

These thymocytes were suspended in an RPMI-1640 complete medium(available from Gibco Corp.), and the numbers of viable cells present inthe suspension were counted by a trypan blue dye exclusion test.

<1-3> Culture of T precursor cells at Various Oxygen Concentrations

The fetal thymocytes (7.0×10⁴ cells/lobe) obtained from the 15-dayfetuses of B6 mice were added to an RPMI-1640 complete medium containingsodium pyruvate, sodium hydrogencarbonate, non-essential amino acids(available from Gibco Corp.), 2-mercaptoethanol, penicillin,streptomycin, and 10% fetal calf serum (FCS) in a 24-wells microtiterplate. The plate was sealed in a plastic vessel (Gaspack available fromBBL Corp or Tedler bag available from Seikagaku Kogyo Inc.), and air inthe vessel was substituted with gas mixtures (available from IwataniSangyo Inc.) respectively containing 5% carbon dioxide and 5% oxygen, 5%carbon dioxide and 20% oxygen, 5% carbon dioxide and 30% oxygen, 5%carbon dioxide and 40% oxygen, 5% carbon dioxide and 60% oxygen, and 5%carbon dioxide and 80% oxygen. The cells were cultured by a submergedculture at 37° C. for 5 days.

In order to compare the present invention with the prior art, fetalthymocytes (7.0×10⁴ cells/lobe) obtained from 15-day fetuses of B6 micewere cultured by an afloat culture under the condition of at a carbondioxide concentration of 5%, an oxygen concentration of 20% and 37° C.for 5 days on a filter (diameter: 13 mm; pore size: 8 μm available fromNucleopore Corp.) floating in an RPMI-1640 complete medium containingsodium pyruvate, sodium hydrogencarbonate, non-essential amino acids(available from Gibco Corp.), 2-mercaptoethanol, penicillin,streptomycin, and a 10% fetal calf serum (FCS) in a 24 well microtiterplate.

After the culture, the numbers of cells were counted by the trypan bluedye exclusion test as in 1-2 above.

Results are summarized in Table 1. In order to examine the relationshipbetween the numbers of recovered viable cells of each population ofcells after the culture and the dissolved oxygen concentration in thenutrient medium, the dissolved oxygen concentrations in the medium aftercompletion of the culture were measured by a blood gas analyzer (StatProfile available from Biomedical Corp.), and results are shown in FIG.1.

                                      TABLE 1                                     __________________________________________________________________________    Gas Phase Concentration                                                       (vol %)       Number of Cells after Culture (× 10.sup.4 /lobe)          O.sub.2  N.sub.2                                                                         CO.sub.2                                                                         Experiment 1                                                                         Experiment 2                                                                         Experiment 3                                                                         Average Value                              __________________________________________________________________________    afloat culture                                                                20       75                                                                              5  --     34.4   32.0   33.2                                       submerged culture                                                              5       90                                                                              5  2.7    --     2.0    2.4                                        20       75                                                                              5  4.7    3.0    3.5    3.7                                        30       65                                                                              5  7.3    --     --     7.3                                        40       55                                                                              5  11.7   6.9    --     9.3                                        60       35                                                                              5  17.5   27.6   25.5   23.5                                       80       15                                                                              5  --     28.5   29.0   28.8                                       __________________________________________________________________________

The cells proliferated up to about 33.2×10⁴ cells/lobe on the fifth dayby the conventional afloat culture, namely, 5-times cell proliferationwas observed in comparison with the state prior to the culture.

On the other hand, in the submerged culture, the cell proliferation onlyup to about 2.4×10⁴ cells/lobe and 3.7×10⁴ cells/lobe were observed atoxygen concentrations of 5% and 20%, respectively. In contrast, thecells significantly proliferated at an oxygen concentration of 40% ormore. At oxygen concentrations of 40%, 60% and 80% respectively,proliferations up to about 9.3×10⁴ cells/lobe, 23.5×10⁴ cells/lobe, and28.8×10⁴ cells/lobe were observed. In particular, the cells multipliedabout 4 times in comparison with the state prior to the culture at theoxygen concentration of 80%. The cell proliferation at the oxygenconcentration of 80% was almost equal to that of the conventionalcumbersome afloat culture which was not suitable for mass production dueto the complexity of the process.

EXAMPLE 2 Differentiation of T precursor cells (Expression ofDifferentiation Antigens)

Following the same procedures as in 1-3 above, air available in a vesselwas substituted with gas mixtures (from Iwatani Sangyo Inc.) containing5% carbon dioxide and 5% oxygen, 5% carbon dioxide and 20% oxygen, 5%carbon dioxide and 40% oxygen, 5% carbon dioxide and 60% oxygen, and 5%carbon dioxide and 80% oxygen. Then, cells were cultured in the vesselusing a submerged culture and differentiation states of the grown cellswere examined.

In order to compare the present invention with the prior art, cells werecultured by an afloat culture following the same procedures as in 1-3above to examine the differentiation states.

Mouse matured T cells were used as a control.

<2-1> Distribution of Cells

The distribution of cells was measured by a flow cytometer (FACScanavailable from Becton Dickinson Corp.) to observe the differentiationstates of the cultured T precursor cells.

Results are shown in FIGS. 2 to 7.

In these experiments, the distribution of cells roughly classified intoblastic lymphoid cells, whose differentiation and maturation have notbeen completed, and small lymphoid cells whose differentiation andmaturation have been completed and have a diameter of about 5 to 10 μmis examined. By this examination of the cell distribution, it is able toobserve the state of the differentiation and maturation of the Tprecursor cells.

Most of the cells in the population of mouse matured T cells (FIG. 2)were observed as small lymphoid cells. Similarly, in a population ofcells (FIG. 4) obtained by culturing mouse fetal thymuses by an afloatculture at an oxygen concentration of 20%, most of the cells wereobserved as small lymphoid cells.

On the other hand, in populations of cells (FIGS. 5 and 6) obtained byculturing mouse fetal thymuses by a submerged culture at oxygenconcentrations of 5% and 20% respectively, most of the cells wereobserved as blastic lymphoid cells similar to the case of a populationof the mouse fetal thymocytes (FIG. 3) immediately after beingcollected. The result showed that the differentiation and maturationwere not achieved.

On the contrary to those results, in a population of cells (FIG. 7)cultured by a submerged culture at an oxygen concentration of 60%, mostof cells were observed as small lymphoid cells similar to the cases of apopulation of mouse matured T cells (FIG. 2) and the population of cells(FIG. 4) cultured by an afloat culture at an oxygen concentration of20%. The result showed that the differentiation and maturation of the Tprecursor cells were achieved.

<2-2> Expression of Differentiation Antigens

Expression level of mouse differentiation antigens CD4 and CD8 weremeasured by an ordinary method using a flow cytometer (FACScan availablefrom Becton Dickinson Corp.) in accordance with a fluoresance-linkedantibody-mediated analysis using monoclonal antibodies for eachdifferentiation antigen of the mouse T cells, in order to observe thedifferentiation level of the T precursor cells after the culture.

The monoclonal antibodies used in this experiment were a phycoerythrin(PE)-labeled anti-mouse CD4 monoclonal antibody (rat hybridoma GK1.5available from Becton Dickinson Corp.) and a fluorescein isothiocyanate(FITC)-labeled anti-mouse CD8 monoclonal antibody (rat hybridoma 53.6.7available from Becton Dickinson Corp.)

Results are shown in Table 2 and FIGS. 8 to 15.

                  TABLE 2                                                         ______________________________________                                                  Persentage of Cells Expressing Differentiation                                Antigens (CD4 and/or CD8 positive)                                  Gas Phase Oxygen                                                                          Persentage to whole viable                                                                    Percentage to blastic                             Concentration (vol %)                                                                     cells           lymphoid cells                                    ______________________________________                                        afloat culture                                                                20          97.9            90.5                                              submerged culture                                                              5          16.7            16.7                                              20          59.3            34.3                                              40          84.1            56.2                                              60          92.6            69.8                                              80          96.4            81.1                                              ______________________________________                                    

Most of cells in the population of mouse matured T cells (FIG. 8) werefound to be CD4⁺ and/or CD8⁺. Further, most of these cells were CD4⁺CD8³⁰ . Similarly, in a population of cells (FIG. 10) obtained byculturing mouse fetal thymocytes by an afloat culture at an oxygenconcentration of 20%, 90% or more cells of total cells were found to beCD4⁺ and/or CD8⁺.

On the other hand, in populations of cells (FIGS. 11 and 12) obtained byculturing mouse fetal thymocytes by a submerged culture at oxygenconcentrations of 5% and 20% respectively, most of the cells were CD4⁻CD8⁻. The cells were, therefore, kept undifferentiated in terms ofphenotypes of the differentiation antigens similar to the case of apopulation of mouse fetal thymocytes (FIG. 9) immediately after beingcollected.

In contrast to these populations of cells, in a population of cells(FIG. 13) cultured by a submerged culture at an oxygen concentration of40%, 80% or more of the total cells were CD4⁺ and/or CD8⁺. Inpopulations of cells (FIGS. 14 and 15) cultured by a submerged cultureat oxygen concentrations of 60% and 80% respectively, 90% or more of thetotal cells were CD4⁺ and/or CD8⁺ similar to the cases of thepopulations of cells (FIG. 8) obtained by culturing the populations ofmouse matured T cells (FIG. 8) and the populations of cells (FIG. 10)cultured by an afloat culture at an oxygen concentration of 20%.Moreover, most of the cells in the populations of cells in FIG. 13 wereCD4⁺ CD8⁺. The results showed that the differentiation and maturation ofthe T precursor cells in terms of phenotypes of the differentiationantigens were achieved.

In addition, the ratio of cells expressing the differentiation antigensin the population of blastic lymphoid cells were increased as the oxygenconcentration was raised in the submerged culture. In the population ofblastic lymphoid cells cultured by submerged culture at oxygenconcentrations of 60% and 80%, 70 to 80% of the total cells expressedthe differentiation antigens.

The above results were similar to the ratio of cells expressingdifferentiation antigens in the population of blastic lymphoid cellsobtained by performing an afloat culture at an oxygen concentration of20%, indicating that the differentiation in terms of phenotypes of thedifferentiation antigens was almost perfectly achieved.

<2-3> Expression of T cell Receptor

At one stage of differentiation and maturation, the T precursor cellsexpress an antigen receptor (T cell receptor; TcR) on the cell membrane.The TcR consists of subunits called α-, β-, γ- and δ chains. TcRassociates with CD3, which is one differentiation antigen serving as amolecule for intracellular signal transduction, thereby forming CD3-TCR.Therefore, by examining the expressions of TcRαβ and TcRγδ and theexpression of CD3, the differentiation level and the degree of thefunctional maturation can be evaluated. The T cells providing arecognition mechanism for foreign antigens are mainly the cellsexpressing TcRαβ. The thymus produces a larger number ofTcRαβ-expressing T cells than of TcRγδ-expressing T cells and suppliesthe peripherary with TcRαβ-expressing T cells.

In this experiment, the expression levels of CD3, TcRαβ and TcRγδ wereexamined following the same procedures as in 2-2 above by usingmonoclonal antibodies for their differentiation antigens; CD3, TcRαβ andTcRγδ respectively.

The monoclonal antibodies used in this experiment were an anti-mouse CD3monoclonal antibody (hamster hybridoma 145-2C11 available from SeikagakuKogyo Inc.), an anti-mouse TcRαβ monoclonal antibody (hamster hybridomaH57-597), and an anti-mouse TcRγδ monoclonal antibody (hamster hybridoma3A10). A fluorescein-labeled rabbit anti-hamster immunoglobulin(available from Caltag Corp.) was used as a secondary antibody.

Results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                                            Percentage of Cells                                                           Expressing Antigens                                                           (Number of Cells Expressing                                       Gas Phase Oxygen                                                                          Antigens; × 10.sup.4)                               Form of Culture                                                                         Concentration (vol %)                                                                       CD3     TcRαβ                                                                     TcRγδ                      ______________________________________                                        Before Culture                                                                          --            2.3     0.3    2.1                                                            (0.16)  (0.02) (0.15)                                 After afloat                                                                            20            55.7    48.8   4.7                                    culture                 (19.9)  (17.4) (1.70)                                 After submerged                                                                          5            45.9    11.3   28.5                                   culture                 (0.96)  (0.24) (0.60)                                           20            49.2    19.3   24.3                                                           (1.70)  (0.68) (0.85)                                           60            63.2    46.9   14.4                                                           (12.6)  (9.40) (2.90)                                 ______________________________________                                    

As for the expression of CD3, ratio of CD3 -positive cells was 63.2% ina population of cells cultured by a submerged culture at an oxygenconcentration of 60%. This expression ratio was equal to or higher thanthe expression ratio of 55.7% in the population of cells cultured by anafloat culture at an oxygen concentration of 20%, thus indicating thatthe differentiation of the T precursor cells was achieved.

The expressions of TcRαβ and TcRy6 occurred as follows. TheTcRαβ-expressing cells and TcRγδ-expressing cells were respectively48.8% and 4.7% in the population of cells cultured by an afloat cultureat an oxygen concentration of 20%. That is, the differentiation of theTcRαβ-expressing T cells occurred selectively.

On the other hand, the TcRαβ-expressing cells were respectively 11.3%and 19.3% in the population of cells cultured by a submerged culture atoxygen concentrations of 5% and 20%, while the TcRγδ-expressing cellswere 28.5% and 24.3% in these populations of cells. The differentiationto TcRγδ-expressing T cells mainly occurred, and the differentiation tothe TcRαβ-expressing T cells was insufficient.

In contrast to these results, the TcRαβ-expressing cells andTcRγδ-expressing cells were respectively 46.9% and 14.4% in a populationof cells cultured by a submerged culture at an oxygen concentration of60%. The differentiation to the TcRαβ-expressing T cells selectivelyoccurred equally to that of the population of cells cultured by theafloat culture at the oxygen concentration of 20%, and indicating thatnormal differentiation of the T precursor cells was achieved.

In addition, the numbers of the recovered cells expressing CD3, TcRαβand TcRγδ respectively, which were generated by the culture under therespective culture conditions were counted. CD3-, TcRαβ-, andTcRγδ-expressing cells were not sufficiently observed in populations ofcells cultured by a submerged culture at oxygen concentrations of 5% and20% respectively. However, CD3-positive and TcRαβ-positive cells weregenerated by 7 to 13 times and by 14 to 39 times, respectively, in apopulation of cells cultured by a submerged culture at an oxygenconcentration of 60%. This result is almost equal to the result in thecase of the population of cells cultured by the afloat culture at theoxygen concentration of 20%.

EXAMPLE 3 Generation of Functional T cells

<3-1> Proliferation Activity of Functional T cells Induced with Mitogen

Substances which activate lymphocytes and induce DNA synthesis and cellproliferation are generically called mitogens (division acceleratingfactor). Concanavalin-A (ConA), one of the mitogens, acts through aCD3-TcR (shown in 2-3) complex which participates in antigen recognitionon the T cells, thereby inducing the proliferation of T cells. Theproliferation of the T cells induced by ConA is maintained by cytokinessuch as Interleukin-2 (IL-2) which is secreted into the culturesupernatant from the T cells upon ConA stimulus.

In this experiment, following the same procedures as in 2-3 describedabove, thymuses were cultured for 5 days at various oxygenconcentrations, using a 24-well microtiter plate. Thus obtained T cellswere cultured in an RPMI-1640 complete medium containing a 10% FCS inthe presence of ConA (15 mg/ml available from Vector Corp.) and IL-2 (50units; available from Genzyme Corp.) using a 96-well microtiter plate,and then, derivation and generation of functionally matured T cellshaving ConA reactivity were examined.

The ConA reactivity of the T cells was determined as follows. Thecultures were performed for 3 days, then, 1.0 μCi of tritium-labeledthymidine (³ H-TdR) was added to each medium at 6 hours before from theend of the culture, and cells were harvested onto glass filters. Then, ascintillator (Clear-SolI available from Nakarai Techs Inc.) was added toeach sample, and intracellular incorporation of the ³ H-TdR was measuredusing a liquid scintillation counter.

Results are shown in FIG. 16.

The proliferation of populations of cells cultured by a submergedculture at oxygen concentrations 5% and 20% were 1/4 to 1/3 respectivelyin comparison with that of a population of cells cultured by an afloatculture at an oxygen concentration of 20%. On the contary, theproliferation of a population of cells cultured by a submerged cultureat an oxygen concentration of 60% was almost equal to that in the caseof a population of cells cultured by an afloat culture at an oxygenconcentration of 20%, showing that the functionally matured T cellshaving the ConA reactivity were derived and generated at the same degreewith the differentiation in the thymuses of young mice and thedifferentiation in the fetal thymuses by the afloat culture.

<3-2> Reactivity of Functional T cells against Allo (BALB/C Mouse)Spleen Cells

T cells are educated in the thymus and acquire reactivity against allomouse cells which are non-self, thereby causing graft rejection, forexample. Similarly to the action of ConA described in 3-1 above, thisreaction is also caused via a TcR-CD3 complex on the T cells which havematured in the thymus.

In this experiment, thymuses were cultured for 6 days at various oxygenconcentrations, using 24-well microtiter plates as in the method in 2-3above. T cells thus obtained were cultured in a 96-well microtiter plate(5×10⁵ cells/well), to which were added cell suspensions which wereobtained by pretreating spleen cells collected from 6-week old BALB/Cmice with mitomycin C (25 μg/ml, 37° C., 30 minutes), subsequentlywashing the treated cells with an RPMI-1640 medium three times. Then,generation of the T cells having reactivity against the allo cells wasexamined.

The allo reactivity of the T cells was determined as follows. Thecultures were performed for 5 days, then, 1.0 μCi of tritium-labeledthymidine (³ H-TdR) was added to each medium at 6 hours before the endof the culture, and cells were harvested onto glass filters. Ascintillator (Clear-SolI available from Nakarai Techs Inc.) was added toeach sample, and intracellular incorporation of the ³ H-TdR was measuredusing a liquid scintillation counter.

Results are shown in FIG. 17.

The proliferation of populations of cells cultured by a submergedculture at oxygen concentrations 5% and 20% were 1/10 to 1/5 comparedwith that of a population of cells cultured by an afloat culture at anoxygen concentration of 20%. In contrast to the results, theproliferation of a population of cells cultured by a submerged cultureat an oxygen concentration of 60% was almost equal to that of apopulation of cells cultured by an afloat culture at an oxygenconcentration of 20%, which indicated that the functionally matured Tcells having the allo reactivity were derived and generated at the samedegree with the differentiation in the thymuses of young mice and thedifferentiation in the fetal thymuses by the afloat culture.

EXAMPLE 4 Differentiation and Maturation of T precursor cells byCombination of T precursor cells and Interstitial Cells of LymphoidTissues

Normal differentiation and maturation of T precursor cells using mousefetal thymuses by applying the technique of the present invention havebeen described in Examples 1 to 3. According to the present invention,furthermore, normal differentiation and maturation of T precursor cellscan be also achieved by using a combination of T precursor cells andinterstitial cells of lymphoid tissues (e.g., thymic epithelial cells,fibroblasts, other interstitial cells of lymphoid tissues, and/orintercellular substances) surrounding the T precursor cells which arerespectively collected from the same or different individuals of thesame or different species. An example is described below.

Thymuses obtained from 15-day fetuses of B6 mice (available from NipponSLC Corp.) were cultured by an afloat culture in RPMI-1640 completemedium containing 1.35 mM 2'-deoxyguanosine (dGuO) and 10% fetal calfserum (FCS) for 6 days, followed by further afloat culture for 12 hoursin the complete medium without dGuO, thereby obtaining thymicinterstitial cells free from blood cells.

The thymic interstitial cells thus obtained were seeded in a 96-wellround bottom plate, and RPMI-1640 complete medium was added to the plate(200 μl/well). Then, T precursor cells (mouse fetal thymocytes) obtainedb the same procedures as in Example 1-2 were added at 30 cells/well or300 cells/well. The plate was sealed in a plastic vessel (Gaspackavailable from BBL Corp.), and air in the vessel was substituted withgas mixture (available from Iwatani Sangyo Inc.) containing 5% of carbondioxide, 60% of oxygen, and 35% of nitrogen. Thereafter, the sampleswere cultured by a submerged culture for 7 days.

After the culture, the recovered cells and the thymic tissues weregently minced with a stainless steel mesh to obtain T cells. In order toobserve the differentiation and maturation degree of T precursor cellsafter the culture, the expression level of CD4 and CD8, which aredifferentiation antigens of the mouse T cells, was measured followingthe same procedures as in Example 2-2.

A culture obtained by culturing only thymic interstitial cells withoutadding the T precursor cells (mouse fetal thymocytes) by a submergedculture at an oxygen concentration of 60% was used as a control, andmouse matured T cells were used for comparison.

Results are shown in FIGS. 18 to 20.

Most of the cells in the population of mouse matured T cells group (FIG.20) were CD4⁺ and/or CD8⁺. Further, most of these cells were CD4⁺ CD8⁺.

In the population of cells (FIG. 18) cultured by a submerged culture atan oxygen concentration of 60% without adding the T precursor cells(mouse fetal thymocytes), very few cells (including thymic epithelialcells, fibroblasts, and so on) were recovered, very few cells exhibitedCD4⁺ and/or CD8⁺.

On the other hand, in the populations of cells obtained from the cultureby a combination of mouse thymic interstitial cells and T precursorcells (mouse fetal thymocytes) (30 or 300 cells each), which wereindividually collected, by a submerged culture at an oxygenconcentration of 60%, a large number of cells were found to be CD4⁺and/or CD8⁺. When 30 cells/well mouse fetal thymocytes were used, 30 to40% cells exhibited CD4⁺ and/or CD8⁺. When 300 cells/well of mouse fetalthymocytes were used (FIG. 19), 90% or more of the cells exhibited CD4⁺CD8⁺.

The result in the culture by combination is equivalent to that in thecase of the population of mouse matured T cells, showing that thedifferentiation and maturation of the T precursor cells in terms oftheir phenotypes of the differentiation antigens were achieved even by aculture with a combination of the mouse fetal thymocytes and the mousethymic interstitial cells which were individually collected.

EXAMPLE 5 Culture of Allogenic T precursor cells in Matured T cellsDeficient Animal Model

When the thymus functions for proliferating, differentiating, andmaturing B precursor cells and/or T precursor cells are deficient or thematured B cells and/or the matured T cells are congenitally deficient,various diseases owing to a deficient immune system are induced. Even ifthe matured B cells or matured T cells were administered in an adoptiveimmunotherapy, matured B cells or matured T cells having an appropriateantigen specificity cannot be induced due to the deficiency of lymphoidprecursor cells (e.g., B precursor cells and T precursor) or theabnormality of the proliferation, differentiation, and maturationprocess in the thymus. Therefore, there is little significance oftherapy. In order to examine the therapeutic significance value of thepresent invention in such a model, the following experiment wasperformed.

Thymuses were aseptically collected from 6-week old SCID mice (severecombined immunodeficient mouse; available from Fox Chase Corp.) whichare the model of mice having defects in abilities of differentiation andmaturation of B precursor cells and T precursor cells, following thesame procedures as in Example 1-1. The obtained each thymus was dividedinto four pieces. Each piece was added to an RPMI-1640 complete mediumusing a 96-well microtiter plate.

Fetal thymocytes as T precursor cells were obtained from 14-day fetusesof C57BL/6 mice (available from Nippon SLC Inc.) following the sameprocedures as in Examples 1-1 and 1-2 and they were added by the numberof 5×10⁴ cells/well into the RPMI-1640 complete medium containing thethymus pieces of the SCID mouse.

The cells in the culture plate were subjected to a submerged culture at37° C. for 7 days, in an atmosphere having a carbon dioxideconcentration of 5% and an oxygen concentration of 20% (normalatmospheric condition) and in an atmosphere having a carbon dioxideconcentration of 5% an oxygen concentration of 80%.

In order to improve efficiency of the differentiation and maturation ofthe T precursor cells, a culture was performed under the same conditionsusing the SCID mouse thymuses which T precursor cells having nodifferentiation or maturation abilities were removed by radiation (2.5Gr).

The number of cells after culture was counted by the trypan blue dyeexclusion test.

The results are shown in Table 4. Each described value is an average ofexperimental values obtained using four-split thymus pieces.

                  TABLE 4                                                         ______________________________________                                        Gas Phase         Number of cells after                                       concentration     culture (cells/lobe)                                        (Vol %)           Radiation-                                                                             Radiation-                                         O.sub.2  N.sub.2                                                                             CO.sub.2   Treated                                                                              Non-Treated                                  ______________________________________                                        20       75    5          --     1.3 × 10.sup.4                         80       15    5          --     2.8 × 10.sup.4                         20       75    5          1.6 × 10.sup.4                                                                 --                                           80       15    5          3.8 × 10.sup.4                                                                 --                                           ______________________________________                                    

In experiments using both radiation-treated thymuses and nontreatedthymuses, populations of cells cultured at an oxygen concentration of80% exhibited twice or more of a survival rate of T precursor cellscompared with that of populations of cells cultured at an oxygenconcentration of 20% (normal atmospheric condition).

In comparison between the populations of cells cultured at an oxygenconcentration of 80% using radiation-treated or nontreated thymuses, thepopulation of cells using the radiation-treated thymuses exhibited amore significant survival rate of T precursor cells than the populationof cells using the nontreated thymuses. Differentiated and matured Tcells were observed in the viable cells.

EXAMPLE 6 Culture of Human T Precursor Cells at High OxygenConcentration

A part of the thymus close to the heart of a 2-year old patientsuffering from a heart disease was removed in a surgical operation. Theobtained human thymus was divided into pieces each having a same weight(about 10 mg). The prepared thymus pieces (5×10⁵ cells/thymus piece)were treated according to the same procedures as in Example 1, followedby a submerged culture at 37° C. for 14 days in an atmosphere having acarbon dioxide concentration of 5% and an oxygen concentration of 20%,as well as in an atmosphere having a carbon dioxide concentration of 5%and an oxygen concentration of 70%.

The numbers of cells after the culture were counted by the trypan bluedye exclusion method.

Results are summarized in Table 5.

                  TABLE 5                                                         ______________________________________                                        Gas Phase                                                                     concentration                                                                 (Vol %)          Number of Cells after                                        O.sub.2                                                                             N.sub.2     CO.sub.2                                                                             Culture (cells/lobe)                                 ______________________________________                                        20    75          5      0.64 × 10.sup.5                                70    15          5      1.50 × 10.sup.5                                ______________________________________                                    

Similar to the results of Example 1 using the mouse thymocytes, in thisexperiment using the human thymocytes, the survival rate of cells in theculture at the oxygen concentration of 70% (high oxygen concentration)was found to be significantly twice or more than with that found at theoxygen concentration of 20% (normal atmospheric condition).

Judging from the results obtained in Examples 5 and 6, the presentinvention enabled derivation of normally matured T cells from thedifferent strain or same strain T precursor cells even in tissues suchas human thymuses and matured T cell-deficient animal model such as SCIDmice. Therefore, it can be given as a conclusion that the presentinvention is highly applicable to research and development of cellvaccines and therapeutic agents for various autoimmune diseases orimmune diseases due to virus infections, such as AIDS (acquiredimmuno-deficiency syndrome).

EXAMPLE 7 Correlation between Gas Phase Oxygen Concentration andLiquid-Phase Dissobed Oxygen Concentration

The following experiment was conducted to examine the correlationbetween the oxygen concentration in the gas phase contacting a nutrientmedium and the dissolved oxygen concentration in the nutrient medium.

A 96-well microtiter plate containing an RPMI-1640 complete mediumcontaining or not containing mouse fetal thymuses (7×10⁴ cells/lobe)obtained in Example 1 was sealed in a plastic vessel (Gaspack availablefrom BBL Corp. or Tedler bag available from Seikagaku Kogyo Inc.). Asshown in FIG. 21, the carbon dioxide concentration was maintainedconstant (5%), and gas mixture (available from Iwatani Sangyo Inc.)having a predetermined constant mixing ratio of the oxygen concentration(%) to the nitrogen concentration (%) was blown from a vent of thevessel to sufficiently substitute the air in the vessel with the gasmixture. Thereafter, the microtiter plate was stood in the vessel at 37°C. for 1 to 5 days.

After the concentrations of carbon dioxide, oxygen, and nitrogen were inequilibrium between the gas phase and the liquid phase, the dissolvedoxygen concentration (mmHg, μM) in the nutrient medium (liquid phase)was measured using an automatic gas analyzer (available from NovaCorp.).

Results are shown in FIG. 21.

When the volume of the gas mixture (gas phase) contacting with thenutrient medium was relatively larger enough than the volume of thenutrient medium (liquid phase), a proportional relationship was obtainedbetween the gas phase oxygen concentration and the liquid phasedissolved oxygen concentration. In addition, when the volume of the gasphase was not relatively large, it was found that a correlation fallingwithin the region (a hatched portion in FIG. 21) below the proportionalline can be established.

As has been in detail described above, according to the presentinvention, T lineage precursor cells are cultured in a nutrient mediumunder the condition that a dissolved oxygen concentration in thenutrient medium is higher than that in the nutrient medium under thenormal atmospheric air, i.e., under the condition that the dissolvedoxygen concentration is raised by means of raising the oxygenconcentration in the gas phase contacting the nutrient medium to 40 vol%through 95 vol %. By applying the method of the invention, not only canT lineage cells be cultured in a large scale in vitro by a submerged(suspension) culture as a general method of culturing cells or tissueswithout using special tools, but also it is possible to realize in vitrothe proliferation process of the T precursor cells and normaldifferentiation and maturation processes of T precursor cells in termsof the phenotypes of the differentiation antigens and their functions.It is also possible, by a submerged (suspension) culture without usingspecial tools, to derive and generate T cells matured in terms of theirphenotypes of differentiation antigens and their functions, which haveappropriate antigen recognition ability tools. Therefore, the presentinvention can contribute to investigate details of differentiation andmaturation mechanism of the T lineage cells.

Furthermore, by using the method of the present invention, T lineageprecursor cells are cultured in a nutrient medium containing an antigenwhich induces infectious diseases such as a hepatitis virus or HIV(human immuno-deficiency virus), allergic diseases, or autoimmunediseases, thereby deriving T cells which recognize the antigen.Therefore, appropriate T-cell vaccines capable of suppressing ortreating the diseases such as infectious diseases, allergic diseases,and autoimmune diseases induced by the antigen can be prepared.

I claim:
 1. A method to produce matured mammalian T cells specific foran antigen which induces infectious diseases, allergic diseases orautoimmune diseases, comprising a step of culturing mammalian Tprecursor cells or mammalian tissue containing mammalian T precursorcells together with mammalian interstitial cells of a thymus in anutrient medium containing said antigen, said nutrient medium having adissolved oxygen concentration ranging from 360 μM to 855 μM orcontacting a gas phase having an oxygen content ranging from 40% to 95%,to obtain said matured T cells having a specificity for said antigen. 2.The method of claim 1, wherein said nutrient medium has a dissolvedoxygen concentration ranging from 540 μM to 855 μM.
 3. The method ofclaim 1, wherein said nutrient medium contacts a gas phase having anoxygen content ranging from 60% to 95%.
 4. The method of claim 1,wherein said mammalian T precursor cells are obtained from a tissue ofan organ selected from the group consisting of thymus, bone marrow andliver.
 5. The method of claim 1, wherein said mammalian tissuecontaining mammalian T precursor cells is obtained from an organselected from the group consisting of thymus, bone marrow and liver. 6.The method of claim 1, wherein said mammalian interstitial cells of athymus and said mammalian T precursor cells or mammalian tissuecontaining mammalian T precursor cells are respectively obtained from asingle individual.
 7. The method of claim 1, wherein said mammalianinterstitial cells of a thymus and said mammalian T precursor cells orsaid mammalian tissue containing mammalian T precursor cells arerespectively obtained from different individuals of the same species. 8.The method of claim 1, wherein said mammalian interstitial cells of athymus and said mammalian T precursor cells or said mammalian tissuecontaining mammalian T precursor cells are respectively obtained fromindividuals of different species.
 9. The method of claim 1, whichfurther comprises collecting the matured T cells so obtained.